Type 2 diabetes is characterized by islet ?-cell failure, contributed to by hyperglycemia and hyperlipidemia. Neprilysin (NEP) is a widely expressed plasma membrane peptidase that we have shown is increased in islets with prolonged exposure to elevated glucose and fat. Like dipeptidyl peptidase-4 (DPP-4), NEP can degrade and inactivate glucagon-like peptide-1 (GLP-1), a glucose-dependent insulinotropic peptide secreted by intestinal L cells and islet ? cells. Our preliminary data show that under conditions of increased dietary fat, NEP ablation in mice enhances active GLP-1 levels thereby contributing to increased glucose-stimulated insulin secretion (GSIS) and improved glucose tolerance. Thus in this proposal, we will test the hypothesis that increased NEP activity limits the beneficial effect of GLP-1 on insulin secretion and glucose homeostasis. To address this hypothesis, the following specific aims have been identified: 1) To determine the contribution of islet NEP to reduced GSIS via its ability to degrade active GLP-1. First, an in vitro culture model will be utilized to measure active GLP-1 levels in human and mouse islets cultured with or without palmitate or high glucose to determine whether GLP-1 levels are reduced in conditions associated with increased NEP activity. Islets with pharmacological inhibition or genetic ablation of NEP will be used for comparison. Second, an in vivo model will be utilized in which wild-type or NEP-/- islets will be transplanted into streptozocin-diabetic wild-type or NEP-/- syngeneic recipients to evaluate the contribution of islet NEP to the maintenance of active GLP-1 levels and normal ?-cell function following transplantation. 2) To determine the contribution of intestinal NEP to reduced GSIS via its ability to degrade active GLP- 1. GLP-1 released from L cells facilitates GSIS directly via the systemic circulation and indirectly via neural signals from vagal afferents. We will assess whether selective reduction of NEP activity in mouse intestine using pharmacologic and genetic (NEPflox ? Villin-Cre) approaches increases portal and peripheral active GLP- 1 levels and thereby enhances GSIS. The contribution of neural signaling to enhanced GSIS will be determined using selective hepatic vagotomies or capsaicin to block activation of pancreatic vagal efferents. 3) To compare the efficacy of NEP versus DPP-4 inhibition in enhancing active GLP-1 levels and improving fat-induced insulin secretory dysfunction in vivo. NEP, DPP-4 or both will be pharmacologically inhibited in wild-type control mice fed a low or high fat diet for 18 weeks. Active GLP-1 levels and GSIS will be measured to determine the relative contributions of each peptidase to preservation of ?-cell function in mice fed a high fat diet. Dual peptidase inhibition will test whether any beneficial effect observed wit NEP inhibition can be augmented with concurrent DPP-4 inhibition. These studies will delineate a novel role for islet and intestinal NEP in modulating ?-cell function, and will have significant implications for development of therapeutics to treat ?-cell dysfunction in diabetes.